WO2017051984A1 - Adenophora triphylla extract containing adenophora triphylla saponins as bile acid regulators or fxr agonists, and pharmaceutical composition or dietary supplement composition for preventing or treating steatohepatitis and primary biliary cirrhosis, containing same - Google Patents

Abstract

The present invention relates to: an Adenophora triphylla extract containing Adenophora triphylla saponins as bile acid regulators or farnesoid X receptor (FXR) agonists; and a pharmaceutical composition or a dietary supplement composition for preventing, alleviating or treating steatohepatitis and primary biliary cirrhosis, containing the same. According to the present invention, the Adenophora triphylla saponins or a composition containing, as an active ingredient, an Adenophora triphylla extract containing the same is harmless to the human body, inhibits fat synthesis by acting as a bile acid regulator or an FXR agonist, and has a remarkable effect of inhibiting neutral fat accumulation within liver tissue by promoting fatty acid oxidation, thereby being usable as an agent or a dietary supplement for preventing, alleviating or treating fatty liver diseases. In addition, the composition of the present invention can play a role in the treatment of general liver diseases such as nonalcoholic steatohepatitis and alcoholic hepatitis and, particularly, has a function of activating and regulating bile acid, an antifibrillogenic function and an antiinflammatory function, thereby being usable as an agent or a dietary supplement for preventing, alleviating or treating primary biliary cirrhosis.

Description

Residual extract containing remnant saponin as a bile acid modulator or FRR anti-inflammatory agent, and a pharmaceutical composition for the prevention or treatment of fatty hepatitis and primary biliary cirrhosis or a composition for functional food

The present invention relates to a bile acid modulator or FXR agonist, and a pharmaceutical composition for the prevention or treatment of fatty hepatitis and primary biliary cirrhosis or a composition for health food containing the same.

The function of bile acids in vivo is known to act as an emulsifier in food intake, regulating mechanisms to restore the absorption of dietary cholesterol and nutrients, inflammation and fibrosis, which can cause damage in the liver, small intestine and kidneys. Therefore, bile acid dysfunction or the production and regulation of bile acids is a critical cause of fatty liver (salt), primary biliary cirrhosis. Nuclear Receptor FXR, which regulates the synthesis and clearance of bile acids in the liver, regulates the growth of excess bile acids to target treatment of PBC (Primary Biliary Cirrhosis) and PSC (Primary Sclerosing Cholangitis). And induces mechanisms for treating common liver diseases such as non-alcoholic hepatitis (NASH) and alcoholic hepatitis. Bile activation of FXR induces antifibrosis, anti-inflammatory, and fat accumulation inhibitory effects. FXR is a potential target for the treatment of digestive and liver diseases such as the small and large intestines and the kidneys. Therefore, developing a bile acid regulator or an FXR agonist or inverse-agonist to develop a fatty liver and cirrhosis treatment, a dietary supplement, and its ingredients are very urgently needed in the treatment and prevention of fatty liver disease. Is important.

Fatty liver disease is a disease in which fat, especially triglycerides accumulate in hepatocytes, accounting for more than 5% of the weight of the liver. Among the triglycerides of increased hepatocytes, 60% of fatty acids are introduced from fatty tissues and 25% of them are in the liver cells. Known as free fatty acids produced by Clinically, fatty liver is classified as fatty liver when more than 5% of hepatocytes are observed, or when more than 5 mg of fat per 100 mg of liver is present. Primary Biliary Cirrhosis (PBC) is the primary rare liver disease that results from autoimmune destruction of the bile ducts that release bile acids out of the liver, and persistent accumulation of bile acids causes chronic inflammation and liver damage. Increased toxicity is used as a diagnostic biomarker for primary biliary cirrhosis due to an increase in alkaline phosphatase (ALP).

The liver, which is the largest organ in the human body and where metabolism is most active, is located between the digestive system and the systemic circulation system, and functions to defend and protect the whole body from in vitro exogenous substances. More importantly, the liver is a very important organ responsible for various metabolism, detoxification, degradation, synthesis and secretion. More specifically, the liver regulates and manages energy metabolism, and the nutrients absorbed from the foods eaten are metabolized into substances that can produce energy in the liver. About 2,000 enzymes, various serum proteins, bile acids, phospholipids, cholesterol and fats are synthesized in the liver and distributed systemically. The liver plays a crucial role in biological activity and life support because of its ability to excrete various metabolites into the duodenum through the bile ducts and its immune function. The most well-known liver function is detoxification and decomposition, and liver cells are easily damaged while detoxifying toxic substances introduced from various foods, drugs, and alcohol, and liver diseases may occur due to various causes. Most of the substances entering the living body once passed through the liver, the liver is exposed to many toxic substances and can be damaged by this.

The liver is the only organ with very good regenerative capacity, and it is known to recover most of the time when there is a slight injury.However, the liver persists due to various causes such as viral infection and excessive accumulation of fat. Inflammation can lead to a chronic condition that can't be restored to normal liver, including decreased liver function. As the liver damage becomes chronic, it gradually progresses to chronic hepatitis, fibrosis and cirrhosis, and liver cancer. Chronic hepatitis, liver fibrosis and cirrhosis, and liver cancer currently lack any clear treatment other than liver transplantation. Therefore, preventing or treating liver damage before it progresses chronically is a very important task that can inhibit or reverse the progression of liver fibrosis, cirrhosis, liver cancer.

These liver damage and liver diseases can be distinguished according to the cause, but the area that is currently the most interest in health and medical care is the area of fatty liver and primary biliary cirrhosis associated with the production and regulation of bile acids. In particular, non-alcoholic steatohepatitis is spreading like an epidemic in developed countries as the time for high calorie meals and sedentary life increases. As the cause of the development and worsening of non-alcoholic base hepatitis (NASH) is increasing, the interest of medical and pharmaceutical companies is increasing.

Fatty liver is divided into alcoholic fatty liver and non-alcoholic fatty liver. Fat accumulates in the liver cells, which crushes the nuclei and other contents of the liver cells, and the scar tissue pushes out healthy liver cells, which leads to cirrhosis or serious liver damage.

Alcohol is the major cause of fatty liver disease, and repeated alcohol intake leads to an increase in NADH and NADP ⁺ during metabolism, and an increase in NADH promotes fatty synthesis, leading to fatty liver.

Nonalcoholic fatty liver disease (NAFLD) is a simple steatosis with only excessive accumulation of fat in the liver cells, nonalcoholic steatohepatitis (NAS) with hepatocellular necrosis, inflammation and fibrosis, and more. A group of diseases, including advanced forms of cirrhosis. Nonalcoholic steatohepatitis (NASH) is commonly known to be associated with obesity, which is literally caused by excessive accumulation of fat in the liver, but has been found to be unrelated to alcohol abuse, which in particular causes numerous serious liver diseases. Non-alcoholic hepatitis (NASH) can destroy the liver, so people with severe symptoms may need to have a liver transplant and sometimes die. Most of these nonalcoholic fatty liver diseases are accompanied by complications such as insulin resistance, obesity, diabetes, hyperlipidemia, and must be treated. However, the current principles of treatment are prescribing diet or exercise therapy and several alternative treatments. to be. Therefore, there is an urgent need for the development of medicines for the prevention, improvement and treatment of fatty liver disease or the development of health functional food materials and the development of health functional foods using the same.

Although various products have been attempted as a therapeutic agent for liver diseases including fatty liver, there are no products that have been proved to be effective and supplementary drugs are used. Diet, weight loss, and adjuvant treatment of diabetes and hyperlipidemia are currently used as standard therapies, but they do not determinate disease progression. Currently prescribed adjuvant drugs include weight loss drugs (e.g. Rimonabant, Orlistat, Sibutramine), insulin resistance improving drugs (Pioglitazone, Rosiglitazone, Metformin, etc.), antioxidants (Vitamine E. Vitamine C, etc.), hyperlipidemia drugs (Statin, Clofibrate, Gemifibrozil, etc.) and liver (intestinal) protectants (UDCA, Silymarine, DDB, etc.) are all, but do not fundamentally prevent the progression of fatty liver disease.

On the other hand, Adenophora Radix is a perennial herbaceous native plant belonging to the Campanulaceae, and the young shoots of early spring are known as “beech” as a representative wild vegetable used for food, but the roots are mainly rooted traditional herbs and foods. It is used as. Root is said to have a similar effect to ginseng called "sasam", and is used in herbal medicine such as expectoration, Jinhae, health, and tonic. It is known for its high content of vitamins A, C and calcium, and its main ingredients are saponin and inulin. However, the remaining saponins of the residue is a situation that is hardly known in detail yet. Therefore, the study of the remnants has been very limited, and most of the studies carried out have been conducted as crude extracts of the remnants. In particular, residues of saponins, gipsogenin, quillaic acid, and the like, which are associated with nuclear receptor FXR, which regulates bile acid production and regulation or bile acid synthesis and clearance in hepatocytes, which is associated with fatty liver disease and primary Pharmaceutical compositions for the prevention and treatment of biliary cirrhosis, materials for dietary supplements, and their compositions have not been studied.

Hereinafter, to describe the prior art related to the composition for the prevention and treatment of liver disease.

Korean Patent Registration No. 10-1501433 (Applicant: Hyung-Jin Park, JBK Natural Medicine Research Institute, Mushmed Co., Ltd.) describes a composition for preventing and treating non-alcoholic fatty liver disease, more specifically, black chokeberry (Aronia melanocarpa) and a composition for the prevention and treatment of non-alcoholic fatty liver disease, characterized in that it comprises a thistle (Silybum marianum) extract.

Korean Patent Publication No. 2013-0119146 (Applicant: Catholic University of Korea Industry-Academic Cooperation Group) describes a composition for the prevention and treatment of fatty liver disease containing mineral water, more specifically, potassium 200 ~ 230mg / L, Sodium 7000-9500 mg / L, calcium 1400-1700 mg / L, magnesium 900-1100 mg / L, zinc 3-9 mg / L, strontium 25-35 mg / L, selenium 200-500 µg / L, vanadium 65 75 μg / L, germanium 0.5-1.5 μg / L, manganese 10-40 μg / L, cobalt 1-3 μg / L, titanium 600-950 μg / L, copper 3-9 μg / L, lithium 0.02-0.09 Mg / L, chlorine ion 16000 ~ 19500mg / L, fluorine ion 1.5 ~ 3.0mg / L, bromine ion 40 ~ 56mg / L, sulfate ion 3000 ~ 4500mg / L, boron 0.8 ~ 1.2mg / L and silicon dioxide A composition for preventing or treating fatty liver disease, including 0.5-15 mg / L, is described.

Republic of Korea Patent Registration No. 10-0953813 (Applicant: Lee Jung-sik) describes a composition for the prevention and treatment of fatty liver disease containing a complex herbal extract having a lipid inhibitory activity as an active ingredient, more specifically, fingering, Hwanggi, Injin And a pharmaceutical composition for the prevention and treatment of alcoholic fatty liver, obese fatty liver or diabetic fatty liver, which contains a complex herbal extract mixed with the extract of Schisandra chinensis as 0.1 to 10: 0.1 to 10: 0.1 to 10: 1 as an active ingredient. It is described.

Korean Patent Registration No. 10-1072247 (Applicant: Univera Co., Ltd., Industry-University Cooperation Group of Sungkyunkwan University) describes processed aloe vera extract for treating or treating liver disease, and a combination therapy of processed aloe vera extract and milk thistle. , More specifically, the extract obtained by extracting the aloe vera leaf with a solvent is concentrated, and the concentrated solution is exposed to 60 to 100 ℃ and then lyophilized and the processed aloe vera extract and milk thistle as an active ingredient The pharmaceutical composition for the prevention or treatment of liver disease, characterized in that the weight ratio of the aloe vera extract and milk thistle is 1: 1.2 to 2.

Korean Patent Registration No. 10-1523663 (Applicant: Korea Institute of Oriental Medicine) describes a composition for the prevention or treatment of fatty liver disease or obesity, including mixed herbal extracts of allies, licorice, ginger and hoo, more specifically Pharmaceutical composition for the prevention or treatment of fatty liver disease or obesity, or fatty liver disease, comprising a mixed herbal extract of Arctium lappa Linne, Licorice (Glycyrrhiza uralensis Fischer), Ginger (Zingiberis rhizoma Crudus), and Magnoliae Cortex Or a food composition for preventing or improving obesity; And a method for treating fatty liver disease or obesity using the pharmaceutical composition.

An object of the present invention is to contain bile acid (Adenophora Radix) saponin or a remnant extract containing the same as an active ingredient, bile acid regulator or FXR (pane to regulate bile acids in liver tissues or inhibit the synthesis of fat and promote fatty acid oxidation Soy X receptor).

In addition, another object of the present invention is to provide a pharmaceutical composition and a functional food composition for the prevention or treatment of fatty liver disease containing the bile acid regulator or FXR agonist.

In order to achieve the above object, the present invention provides a bile acid modulator or FXR (panesoid X receptor) adjuvant containing a residual saponin or a residual extract containing the same as an active ingredient.

In one embodiment, the residue extract is preferably a hot water extract or an organic solvent extract.

In one embodiment, the residue hydrothermal extract is obtained by drying and grinding the residue in the shade to obtain a residue powder; Adding 5-10 times water by volume to the obtained residue powder and extracting twice for 4 to 6 hours at a temperature of 90 to 100 ° C. to obtain a hydrothermal extract; And removing the solids by filtration or centrifugation of the hydrothermal extract obtained, and concentrating and drying to obtain a residual hydrothermal extract powder.

In one embodiment, the residue organic solvent extract is dried and pulverized the residue in the shade to obtain a residue powder; To the obtained residue powder, an alcohol extract was obtained by adding 1 to 5 times C ₁ to C ₄ alcohol, or a mixture of C ₁ to C ₄ alcohol and water, and extracting twice at a temperature of 90 to 95 ° C. for 3 hours. Obtaining; And the obtained alcohol extract is filtered or centrifuged to remove solids, concentrated and dried to obtain a residual alcohol extract powder.

In one embodiment, the C ₁ ~ C ₄ alcohol is ethanol, the alcohol extract is an ethanol extract, the residue alcohol extract powder is preferably a residue ethanol extract powder.

In one embodiment, the residue extract preferably comprises 5 to 100% by weight.

In addition, the present invention provides a pharmaceutical composition and a functional food composition for preventing and treating fatty liver disease containing the bile acid regulator or FXR agonist as an active ingredient.

In one embodiment, the fatty liver disease is preferably any one selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, obese fatty liver, and fatty liver disease group of diabetic fatty liver and primary biliary cirrhosis.

The composition containing the remnant saponin or the remnant extract containing the same as an active ingredient is harmless to the human body and acts as a bile acid regulator or FXR (panesoid X receptor) adjuvant to inhibit the synthesis of fat and promote fatty acid oxidation. Since the effect of inhibiting triglyceride accumulation in the liver tissue is excellent, it can be usefully used as a preventive and therapeutic agent for fatty liver disease or health functional food. In addition, the composition of the present invention may play a role in treating common liver diseases such as nonalcoholic steatohepatitis and alcoholic hepatitis, and in particular, has a function of bile activation and regulation, and has an antifibrotic function and an anti-inflammatory function, thereby causing primary biliary cirrhosis. It can be usefully used as a preventive and therapeutic agent or as a dietary supplement.

1 is a graph showing the toxicity evaluation in the hepatocytes of the extract according to Example 1.

Figure 2 is a diagram showing the inhibitory effect of SREBP-1c, FAS gene expression on the mRNA level of the residue extract according to Example 1.

3 and 4 are graphs showing the effect of inhibiting fat accumulation in hepatocytes of the extract according to Example 1 (FIG. 3: 8 hours treatment, FIG. 4: 24 hours treatment).

5 is a graph showing the inhibitory effect of ROS expression in hepatocytes of the extract according to Example 1.

Figure 6 is a view showing the SREBP-1c, FAS gene expression inhibitory effect on the protein level of the remnant extract according to Example 1.

Figure 7 is a graph showing the weight change evaluation of the nonalcoholic steatohepatitis animal model of the residue according to Example 1.

Figure 8 is a graph showing the evaluation of blood glucose changes in the nonalcoholic steatohepatitis animal model of the residue according to Example 1.

Figure 9 is a graph showing the inhibitory effect of cholesterol accumulation in the nonalcoholic steatohepatitis animal model of the extract according to Example 1.

10 is a graph showing the evaluation of triglyceride accumulation in the nonalcoholic steatohepatitis animal model of the residual extract according to Example 1.

11 and 12 are diagrams showing the pathological observation evaluation of the nonalcoholic steatohepatitis animal model of the extract according to Example 1.

Figure 13 is a graph showing the effect of inhibiting the expression of ALT, AST in the nonalcoholic steatohepatitis animal model of the extract according to Example 1.

14 is a graph showing the effect of inhibiting the expression of TGF-β in the nonalcoholic steatohepatitis animal model of the extract according to Example 1.

15 is a SREBP-1c, FAS AMPK expression inhibition effect in the nonalcoholic steatohepatitis animal model of the extract according to Example 1.

Figure 16 is a graph showing the inhibition of Hedgehog signal that causes fibrosis of liver tissue of the remnant extract according to Example 1.

Unless defined otherwise, all technical terms used in the present invention have the following definitions and conform to the meanings that are commonly understood by those of ordinary skill in the art and those skilled in the art. Also described herein are preferred methods or samples, but similar or equivalent ones are within the scope of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

The term "about" means 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, by reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. By amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by 3, 2 or 1%.

Throughout the specification, the terms “comprises” and “comprising” include, but are not limited to, any other step or component, or step provided, unless the context requires otherwise. Or it is to be understood that it does not exclude a group of components.

In the present invention, 'panesoid X receptor (FXR)' is one of the nuclear receptors closely related to fatty liver disease. FXR is activated when bile acids bind, and FXR promotes the expression of apolipoprotein CII (ApoCII) and inhibits the expression of ApoCIII, thereby increasing the activity of lipoprotein lipase, thereby increasing triglycerides in the blood (triglycerides). ), Goodwin B et al., Mol. Cell, 6, 517-526 (2000); Claudel T et al., Gastroenterology, 125, 544-555 (2003). In addition, it inhibits the expression of sterol-response-element-binding protein 1c (sterol-response-component-binding protein 1c; SREBP1c) and fatty acid synthase (fatty acid synthase; FAS) in the liver, thereby reducing triglyceride synthesis in the liver. (See Watanabe M et al., J. Clin. Invest. 113, 1408-1418 (2004)], promote the expression of PPARα to increase fatty acid oxidation (Jorg A et al., Journal of Lipids, 2012, 1-10 (2012)).

As used herein, the term "improvement" refers to any action by which administration of the composition of the present invention improves or beneficially alters the symptoms of fatty liver disease.

As used herein, the term "administration" means providing a subject with a composition of the present invention in any suitable manner.

As used herein, the term "individual" means any animal, such as a human, monkey, dog, goat, pig, or rat, having a disease in which the symptoms of fatty liver disease can be improved by administering the composition of the present invention.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which means the type of disease, the severity, the activity of the drug, the drug Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention provides a bile acid modulator or FXR (panesoid X receptor) adjuvant containing a residual saponin or a residual extract including the same as an active ingredient.

In the present invention, the residual saponin is to include quillaic acid (Formula 1 (a)) and gipsogenin (Formula 1 (b)), as represented by the following formula [1] , Minseok Kang et al., Phytochem. Anal. 24, 148-154 (2012).

[Formula 1]

Such compounds may be purchased as commercially available materials, prepared by organic chemical methods according to known literature, or obtained by extracting from plants, more particularly residues, without particular limitation.

As an example, a method of obtaining Chemical Formula 1 from the residue is as follows.

Adding a mixture of 90% methanol and hexane to the dried residue powder and extracting at room temperature to obtain a 90% methanol layer and a hexane layer (step 1); This methanol layer was added water, 30-100% methanol, which was separated by a column (e.g., a Dianion HP-20 resin column) to obtain a water fraction and a 30% to 100% methanol fraction, respectively. Step 2); And performing column chromatography (HSCCC) on 70% methanol fractions of these fractions to obtain compounds of Formula 1, respectively (Step 3) [Minseok Kang et al ., Phytochem. Anal. 24, 148-154 (2012).

The residual saponins used in the present invention include not only the above-mentioned substance itself, but also all pharmaceutically acceptable salts thereof, as well as isomers thereof or possible solvates or hydrates that can be prepared therefrom.

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate.

The acid addition salts according to the invention are dissolved in conventional methods, for example, by dissolving a compound of formula 1 in an excess of aqueous acid solution and using the water miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation. It may also be prepared by evaporating the solvent or excess acid from the mixture and then drying or by suction filtration of the precipitated salt.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

In addition, as another active ingredient of the present invention, it may include a residue extract containing the residual saponin represented by the formula (1).

In the present invention, the Adenophora Radix extract may be obtained by extraction and separation from nature using extraction and separation methods known in the art, and the 'extract' defined in the present invention may be prepared by using an appropriate solvent. It is extracted from the residue and may include, for example, all of the hydrothermal extract, polar solvent soluble extract, or nonpolar solvent soluble extract of the residue.

As a suitable extraction solvent for the residue extract, any solvent that is acceptable in the art may be used, and water or an organic solvent may be used. For example, alcohol having 1 to 4 carbon atoms, acetone, ether including purified water, methanol, ethanol, propanol, isopropanol, butanol, etc. , Solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. It is not limited.

As the extraction method, any one of hot water extraction method, cold leaching extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used. In addition, the desired extract may further be subjected to a conventional fractionation process, it may be purified using conventional purification methods. There is no limitation on the method for preparing the residue extract of the present invention, and any known method may be used.

For example, the residue extract included in the composition of the present invention may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying, which are extracted by the hot water extraction or the solvent extraction method. . In addition, the primary extract is further purified using a variety of chromatography, such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. You can also get Therefore, in the present invention, the residual extract is a concept including all the extracts, fractions and purified products obtained in each step of extraction, fractionation or purification, their dilutions, concentrates or dried products.

In a preferred embodiment, the residue extract may be prepared by solvent extraction of raw residue, dry residue or powder milled thereto. The residue is not particularly limited to the type and production area, and includes both diploid and tetraploid. Preferably, a dry residue may be used, and a drying method may include freeze drying (FD), indoor drying (ID), hot air drying (HD), microwave drying (MD), and the like. As the solvent, ethanol (alcohol) of 0.1 to 100% concentration may be preferably used.

In a specific example, the residue powder is dried in the shade using finely pulverized residue and the hydrothermal extract is added to the residue powder by adding about 2 to 10 times, preferably 5 to 10 times water by volume and 80 to 100 ℃ Preferably, the extract obtained by extracting twice for 4 to 6 hours at a temperature of 90 to 100 ° C. is filtered or centrifuged to remove solids and concentrated, followed by freeze drying and spray drying to completely remove moisture. You can get the frozen residue extract.

In another example, the organic solvent extract is 1 to 5 times, preferably about 3 times, organic solvent, preferably C ₁ to C ₄ alcohol, or C _{1 to} C ₄ alcohol and water, based on the volume of the residual powder. The mixture obtained by adding and then extracting twice at a temperature of 90-100 ° C., preferably 90-95 ° C. for 4-6 hours, preferably 3 hours, was filtered or centrifuged to remove solids, concentrated and freeze-dried. By drying by spray drying, etc., a residue extract from which moisture is completely removed can be obtained. Ethanol may be preferably used as the organic solvent.

Such a residue extract of the present invention may be used alone as a bile acid regulator or FXR agonist, or may be used in admixture with other conventionally known bile acid regulators or FXR agonists or other active ingredients, preferably the residual extract of the present invention may be used as a bile acid regulator or It may comprise from 5 to 100% by weight in the FXR agonist.

The present inventors confirmed that the extract of the remnant has no cytotoxicity, acts as a bile acid regulator or FXR anti-inflammatory agent, inhibits the synthesis of fat, promotes fatty acid oxidation, and inhibits triglyceride accumulation in liver tissue. Accordingly, the compounds of the formula and the residue extracts comprising the same are useful as bile acid modulators or FXR agonists.

In addition, another aspect of the present invention provides a pharmaceutical composition for preventing and treating fatty liver disease containing the bile acid regulator or FXR anti-inflammatory agent of the present invention as an active ingredient.

The pharmaceutical composition of the present invention acts as a bile acid regulator or an FXR agonist to inhibit the synthesis of fat and promote fatty acid oxidation, thereby showing an excellent effect of inhibiting the accumulation of triglycerides in liver tissue, thus showing the prevention and treatment of fatty liver disease.

The composition containing the compound of Formula 1 of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above components.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose , Mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opiodry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , Sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition of 0.1 to 90 parts by weight, but is not limited thereto.

That is, the composition of the present invention can be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used It can be prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate (Calcium carbonate) in the bile acid regulator or FXR agonist of the present invention. ), Sucrose (Sucrose), lactose (Lactose) or gelatin can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally according to a desired method, and when administered parenterally, external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method It is preferable to select. Dosage ranges depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the daily dosage is the amount of active ingredient It can range from 0.02 to 1000 mg / kg body weight, preferably 1 to 200 mg / kg body weight, and may be administered once or several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using other therapies such as procedures, chemotherapy and biological response modifiers for the improvement of fatty liver disease.

The present invention also provides a method for ameliorating fatty liver disease comprising administering to a subject suffering from fatty liver disease a composition containing a pharmaceutically effective amount of a bile acid modulator or FXR agonist of the present invention as an active ingredient.

The present invention also provides a method for improving fatty liver disease, comprising administering to a subject a composition containing a pharmaceutically effective amount of a bile acid modulator or FXR agonist of the present invention as an active ingredient.

The pharmaceutically effective amount is 0.02 to 1000 mg / kg, preferably 1 to 200 mg / kg, but is not limited thereto. The dosage may vary depending on the weight, age, sex, health condition, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of the particular patient.

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably an ape-like animal such as a chimpanzee or gorilla.

The method of administration may be administered orally or parenterally, intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, intrauterine dural, intracerebroventricular or intrathoracic It can be administered by injection.

The bansa saponin and the bansa extract of the present invention acts as a bile acid regulator or FXR agonist to inhibit the synthesis of fat and promote fatty acid oxidation to inhibit triglyceride accumulation in the liver tissues, thereby administering to a subject. It can be usefully used as a method for improving fatty liver disease.

The present invention also provides a food composition for preventing or ameliorating fatty liver disease, which contains a residual saponin or a residual extract including the same as an active ingredient.

Since the saponin and the stalk extract of the present invention acts as a bile acid regulator or FXR agonist, inhibits the synthesis of fat and promotes fatty acid oxidation, thereby preventing the accumulation of triglycerides in liver tissues, thereby preventing and improving fatty liver disease. It can be usefully used as an active ingredient.

Examples of the food to which the saponin of the present invention or the extract of the same containing the same can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of improving fatty liver disease. At this time, the amount of the extract in the food or beverage can be added to 0.01 to 50% by weight, preferably 1 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5g, preferably based on 100 ml It may be added in a ratio of 0.3 to 3 g.

The health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the above-mentioned saponin or the above-mentioned extract as essential ingredients in the indicated ratios, and additional ingredients such as various flavors or natural carbohydrates, such as ordinary drinks. It may contain as. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the residue saponins of the present invention or the residue extracts containing the same include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents such as flavoring agents, colorants and neutralizing agents (cheese, chocolate, etc.), pectic acid And salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.

In addition, it is appropriate to administer the residual saponin of the present invention or the residual extract containing the same, so that the intake amount per adult 1 to 3,000 mg. The dosage can be appropriately increased or decreased depending on age, symptoms, and the like.

Hereinafter, the present invention will be described in more detail based on the preferred embodiments. However, the following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

< Example  1> Preparation of Straw Extract of the Present Invention

(1) Preparation of the residual hydrothermal extract of the present invention

2 L of distilled water was added to 0.2 kg of the residue powder, extracted twice at 90 to 100 ° C. for 5 hours, filtered, and centrifuged (10,000 × g) for 20 minutes to remove solids. This was concentrated using a vacuum distillation and freeze-dried to obtain 100 g of a residual hydrothermal extract in powder form.

(2) Preparation of Residual Ethanol Extract of the Present Invention

3 L of ethanol was added to 1 kg of the residue powder, extracted twice at 90 to 95 ° C. for 3 hours, filtered, and centrifuged (10,000 × g) for 20 minutes to remove solids. This was concentrated using a vacuum distillation and freeze-dried to obtain 150 g of ethanol extract of the powder form.

< Experimental Example  1> Cytotoxicity evaluation of the stalk extract of the present invention

In order to evaluate the cytotoxicity of the residual hydrothermal extract and ethanol extract of the present invention, MTT experiment using hepG2, a hepatocyte cell line, was performed.

To determine cell growth was measured using Cell Counting Kit-8. The medium used for cell culture was used by adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (penicillin-streptomycin) to Dulbecco's Modified Eagle Medium (DMEM). Trypsin-EDTA (GIBCO, USA) and phosphate buffer (PBS) were used for cell division. HepG2 cells were used in this experiment and dispensed into 96 well plates at 5 × 10 ³ cells / ml, respectively. CCK-8 solution was added 1/10 of the culture solution on 1, 3 and 5 days, and then incubated in a 37 ° C. incubator for 2 hours. 100 μl of the cultured solution was dispensed into 96 wells and the absorbance was measured at 450 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, Calif., USA). It was confirmed that the residual hydrothermal extract and residual ethanol extract of Example 1 were not cytotoxic to HepG2 cells (see FIG. 1).

< Experimental Example  2> of twigs extract On FXR  About Antitussives  Active evaluation

In order to elucidate the mechanism of action of the hepatic hydrothermal extract and ethanol extract of the present invention on the efficacy of fatty liver treatment, the panenoid X reporter expresses the antifungal activity against the panesoid X receptor which promotes fatty acid oxidation and inhibits fatty acid synthesis. It was confirmed through a luciferase assay experiment using a cell line (Indigo bioscience).

Panesoid X reporter expressing cell lines stored at −80 ° C. were thawed using cell recovery medium and dispensed into 32-well plates. Residual hot water extract and ethanol extract were diluted with compound screening media and treated with cells. After 24 hours of incubation at 37 ° C. and 5% CO ₂ , the medium was discarded and the Luc-detection solution was aliquoted and left at room temperature for 10 minutes. Agonist activity for the receptor of X Ipanema small beads treated material is to measure the luminescence as a photometric analyzer was determined the EC ₅₀ value, results are shown in Table 1. As a positive control, GW 4064 (sigma) substance, which is an antigen of Panesoid X receptor, was used. The anti-inflammatory activity of the residual hot water extract and ethanol extract against the Panesoid X receptor was confirmed.

TABLE 1

< Experimental Example  3> Sterol-response-component-binding protein 1c involved in fat synthesis SREBP -1c), Fatty Acid Synthase (FAS) Gene Expression Inhibitory Effect

Inhibition of the expression of sterol-response-component-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS) genes involved in fat synthesis in order to elucidate the mechanism of action of fatty acid extract and ethanol extract of the present invention The effect was confirmed by RT-PCR experiment using hepG2, a hepatocyte cell line.

HepG2 cell line contains 37%, 5% CO in a T-75 flask with 10% FBS, penicillin (100 units / ml), streptomycin (100 μg / ml) and sodium bicarbonate (3.7 g / L) in DMEM medium. _The culture was carried out in _two states, and was used for the experiment when 70 to 80% of the culture was performed. The cultured cells were aliquoted into 6-well plates and cultured at 37 ° C. and 5% CO _{2 for} at least 16 hours until the cells formed well. After treatment with the residual hydrothermal extract and ethanol extract was incubated for 24 hours at 37 ℃, 5% CO ₂ state.

After incubation, total RNA was isolated from the cells using a SV total RNA isolation system (Promega corporation) and RNA was quantified using GeneQuant pro (Amersham bioscience). Gene expression was achieved by RT-PCR method using the AccessQuick ™ RT-PCR system (Promega corporation) using primers targeting sterol-response-component-binding protein 1c (SREBP-1c), a fatty acid synthase (FAS) gene. Measured. The negative control used only media solution. Residual hot water extract and ethanol extract were found to inhibit sterol-response-component-binding protein 1c (SREBP-1c), fatty acid synthase (FAS) gene expression in a concentration-dependent manner (see FIG. 2).

< Experimental Example  4> involved in fatty acid oxidation PPARα  Gene expression increase effect

In order to elucidate the mechanism of action of fatty acid extract and ethanol extract of the present invention, the effect of increasing PPARα gene expression involved in fatty acid oxidation was confirmed by RT-PCR experiment using hepG2, a hepatocyte cell line.

HepG2 cell line contains 37%, 5% CO in a T-75 flask with 10% FBS, penicillin (100 units / ml), streptomycin (100 μg / ml) and sodium bicarbonate (3.7 g / L) in DMEM medium. _The culture was carried out in _two states, and was used for the experiment when 70 to 80% of the culture was performed. The cultured cells were aliquoted into 6-well plates and cultured at 37 ° C. and 5% CO _{2 for} at least 16 hours until the cells formed well. After treatment with the residual hydrothermal extract and ethanol extract was incubated for 24 hours at 37 ℃, 5% CO ₂ state.

After incubation, total RNA was isolated from the cells using a SV total RNA isolation system (Promega corporation) and RNA was quantified using GeneQuant pro (Amersham bioscience). Gene expression was measured by RT-PCR method using AccessQuick ™ RT-PCR system (Promega corporation) using primers targeting PPARα gene. The negative control used only media solution. It was confirmed that the residual hydrothermal extract and ethanol extract increased PPARα gene expression in a concentration-dependent manner (see FIG. 3).

< Experimental Example  5> Inhibition of fat accumulation in hepatocytes

In order to determine the efficacy of the hepatic hepatic stem extract and ethanol extract of the present invention, inhibition of fat accumulation in hep cell line HepG2 was confirmed through experiments using a triglyceride colorimetric assay kit (Cayman chemical).

HepG2 cell line contains 37%, 5% CO in a T-75 flask with 10% FBS, penicillin (100 units / ml), streptomycin (100 μg / ml) and sodium bicarbonate (3.7 g / L) in DMEM medium. _The culture was carried out in _two states, and was used for the experiment when 70 to 80% of the culture was performed. The cultured cells were aliquoted into 6-well plates and cultured at 37 ° C. and 5% CO _{2 for} at least 16 hours until the cells formed well. 0.5 mM free fatty acid mixture prepared by dissolving the oleate and palmitate in a molar concentration of 2: 1 was added to the medium, and then treated with a residual hydrothermal extract and an ethanol extract for 8 hours, 24 hours, 37 ° C., Incubated at 5% CO ₂ .

After culturing was completed, the cells were washed and cell lysates were prepared using lysis buffer. After adding the enzyme buffer solution to the cell lysate, the absorbance was measured by ELISA analyzer to analyze the degree of fat accumulation. The negative control used only media solution. It was confirmed that the residual hot water extract and the ethanol extract inhibit the fat accumulation in liver cells excellently (see FIG. 4).

< Experimental Example  6> Determination of Reactive Oxygen Species Changes in Hepatocytes of Straw Extracts

Active oxygen species (reactive oxygen species, ROS) were measured in order to confirm the inhibitory effect of free radical production in the hepatocyte cell line of the present invention.

HepG2 cell line contains 37%, 5% CO in a T-75 flask with 10% FBS, penicillin (100 units / ml), streptomycin (100 μg / ml) and sodium bicarbonate (3.7 g / L) in DMEM medium. _The culture was carried out in _two states, and was used for the experiment when 70 to 80% of the culture was performed. The cultured cells were aliquoted into a 100 mm dish and cultured at 37 ° C. and 5% CO _{2 for} at least 16 hours until the cells formed well. After treatment with the residual hydrothermal extract and ethanol extract was incubated for 24 hours at 37 ℃, 5% CO ₂ state.

Residual hot water extract was removed 12 hours after administration of HepG2 cells at a concentration of 200 μg / ml. The removed residual hydrothermal extract treated HepG2 cells were added 2 ', 7'-dichlorofluorescein diacetate (Sigma-Aldrich, U.S.A) in PBS solution at 30 mM and incubated in a 37 ° C incubator for 30 minutes. The cultured cells were harvested in a 1.5 ml tube and then spun for 10 minutes at 13,000 rpm using a centrifuge. The supernatant was discarded and 300 µl of fresh PBS was added and dissolved in an ultrasonic cleaner for 5 minutes. Once again use a centrifuge for 10 minutes at 13,000 rpm, then dispense 200 μl of the supernatant into a 96 well plate and place it between 485 nm and 530 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). Absorbance was measured. It was confirmed that the reactive oxygen species (ROS) levels were suppressed in the residual hydrothermal extract and the ethanol extract (see FIG. 5).

< Experimental Example  7> of twigs extract Non-alcoholic  Fatty liver metabolism regulation and mechanism study (In vitro)

Sterol regulatory component-binding protein which is a transcription factor that regulates fat metabolism including fatty acid synthase (FAS) in HepG2 hepatocytes in order to elucidate the mechanism of regulating fat synthase transcription of hepatic hydrothermal extract and ethanol extract of the present invention 1 (SREBP-1) and expression for AMPK that inhibits the expression of FAS and SREBP-1c were confirmed using Western blot.

HepG2 cell line was added to DMEM medium with 10% FBS, penicillin (100 units / ml), streptomycin (100 μg / ml) and sodium bicarbonate (3.7 g / L) at 37 ° C, 5% CO2 in a T-75 flask. Cultured in the state, it was used in the experiment when 70 to 80% culture. The cultured cells were aliquoted into 6-well plates and cultured at 37 ° C. and 5% CO _{2 for} at least 16 hours until the cells formed well. After treatment with the residual hydrothermal extract and ethanol extract was incubated for 24 hours at 37 ℃, 5% CO ₂ state. The cultured cells were harvested in a 1.5 ml tube, and then spun for 10 minutes at 13,000 rpm using a centrifuge, and then discarded the supernatant and pellets. Treated with IPH buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 100 μM PMSF), 1 μg / mL protease inhibitors (Luepetin, Aprotinin), 1 mM DTT Protein was extracted. The extracted protein was transferred to a Hybond-P + polyvinylidene difluoride membrane (GE Healthcare, UK) after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membrane was blocked by treatment for 5 hours at room temperature with 5% fat-free milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween-20 and the indicated specific primary antibodies (SREBP-1c and phospho-AMPK). Incubated overnight at 4 ° C with 1% BSA and 0.05% Tween-20 containing TBS. Membranes were further treated at room temperature for 2 hours with peroxidase-conjugated goat anti-mouse antibody or peroxidase-conjugated goat anti-rabbit antibody diluted 1: 3000 again, 0.1% BSA and 0.1% Tween- After washing with 20 containing TBS, protein expression was confirmed using ECL plus (Amersham Biosciences). As a result, it was confirmed that the residual hydrothermal extract and the ethanol extract inhibit the sterol-response-component-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS) gene expression in a concentration-dependent manner (see FIG. 6).

< Experimental Example  8> Straw Hydrothermal  Body Weight and Blood Glucose Measurement in Mice Administered orally with Extract

Body weight and blood glucose of ICR mice, which are animal models, were measured and examined to determine the efficacy of the hepatic hydrothermal extract and ethanol extract of the present invention.

Changes in body weight, starting from the beginning of the diet, using oral administration of 20 mg / kg and 200 mg / kg of residual hot water extract using the Normal Diet and the High Fat Diet, respectively. During the four weeks was measured twice a week while paying.

Blood glucose was measured by a blood glucose meter after blood was taken from the tail vein after 12 hours of fasting of the animals.

Residual hot water extract and ethanol extract was found to decrease the weight and blood glucose levels in a concentration-dependent manner (see FIGS. 7 and 8).

< Experimental Example  9> straw Hydrothermal  Cholesterol and Oral Extracts of Mice Administered Orally Triglycerides  Measure evaluation

Hepatic triglyceride (TG) and total cholesterol content of ICR mice, an animal model, were measured and examined in order to investigate the efficacy of nonalcoholic steatohepatitis disease improvement of the residual hydrothermal extract and ethanol extract of the present invention.

 (1) measuring total cholesterol

Blood was collected using a heparin-treated capillary tube from the tail vein of mice fed oral administration of the residual hydrothermal extract by the Enzymatic Method (Enzymaticmethod). After standing for one hour at, centrifuged at 3000 rpm for 15 minutes to obtain a serum. 300 µl of the enzyme reagent was added to 4 µl of the separated serum and reacted in a 37 ° C. water bath for 15 minutes, and the absorbance (OD) was measured at 550 nm with distilled water as a blank using 96 plate wells. Total cholesterol was measured using the following equation using absorbance of distilled water and the sample.

[Equation 1]

Serum cholesterol (mg / 100ml) = 300 × (absorbance of sample / absorbance of standard)

Residual hot water extract and ethanol extract was confirmed to decrease the total cholesterol level in a concentration-dependent manner (see Figure 9).

(2) triglyceride (TG) measurement

Blood was collected using a heparin-treated capillary from the tail vein of the mouse that was orally administered the remnant hot water extract using a triglyceride kit (Trinder method), and the collected blood was collected for one hour at room temperature. After being left for a while, serum was obtained by centrifugation at 3000 rpm for 15 minutes. 300 μl of the enzyme reagent was added to 4 μl of the separated serum and reacted for 10 minutes in a 37 ° C. water bath. Then, the absorbance (OD) was measured at 500 nm with distilled water as a blank in 1 hour using a 96-well plate. Triglyceride (TG) was measured using the following equation using absorbance of distilled water and the sample.

[Equation 2]

Triglyceride (mg / 100ml) = (absorbance of the sample / absorbance of the standard) × concentration of the standard

The residual hydrothermal extract and ethanol extract was found to decrease triglyceride levels in a concentration-dependent manner (see FIG. 10).

< Experimental Example  10> Histological Observation of Fat Accumulation Inhibitory Effect in the Mouse Liver Tissue (H & E Staining, MT Staining)

In order to elucidate the efficacy of the hepatic hydrothermal extract and ethanol extract of the present invention, the inhibition of fat accumulation was confirmed by experiments using H & E and MT staining in the liver model of ICR mice.

Four-week-old ICR mice were administered high-fat diets for four weeks, and then weighed to select mice that gained weight. Selected mice were divided into three groups (positive control: high fat diet (HFD), HFD + residue extract 20 mg / kg, HFD + residue extract 200 mg / kg), followed by oral administration of the residual hydrothermal extract at a constant time every day for 4 weeks. Five animals were used for each group.

Liver tissues were extracted by sacrificing animals on Days 1, 14, 28, and 56 after oral administration of the residual hydrothermal extract, fixed in 10% neutral formalin solution for 24 hours, and liver tissues were taken and formatted as paraffin after dehydration. The tissue was sliced to a thickness of 5 μm using a tissue slicer and attached to a slide to remove paraffin and water, followed by hematoxylin-eosin (H & E) staining, and Masson's trichrome (MT). ) And oil red O staining. Epidermal growth, inflammatory response, neo-vascular proliferation and collagen deposition were observed using liver tissue.

(1) H & E Dyeing

After removing the paraffin with xylene, the tissue sections were hydrated with 100, 90, 80, 70% ethanol and distilled water for 5 minutes, and washed with distilled water. Stained with Harris hematozain for 3 minutes, the tissue was washed with distilled water for 5 minutes. The washed tissues were stained with eosin for 5 minutes, dehydrated with 70, 80, 90 and 100% ethanol and xylene and then sealed with Shandon Synthetic Mountant (Thermo scientific, USA).

As a result of the experiment, fatty liver findings were observed in the HFD group, but it was confirmed that fatty liver findings were significantly decreased in the remnant hydrothermal extract and ethanol extract administration groups (see FIG. 11).

(2) MT staining

The tissue sections were paraffin-free with xylene and then watered with 100, 90, 80 and 70% ethanol and distilled water for 5 minutes, and washed with distilled water. The tissues were reacted in a Bouin's (IMEB, USA) solution at 60 ° C. for 1 hour and then washed with distilled water. After the reaction and washing, the tissues were treated with Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue coloring solution (IMEB, USA) for 5 minutes and washed with distilled water. After dehydration using 70, 80, 90, 100% ethanol and xylene, it was sealed with Shandon Synthetic Mountant (Thermo scientific, USA).

As a result of the experiment, fibrosis was observed in the HFD group, but it was confirmed that the fibrosis was significantly reduced in the remnant hydrothermal extract and ethanol extract administration groups (see FIG. 12).

< Experimental Example  11> Serum ALT (s-GPT) in mouse liver tissue AST  (s-GOT) enzyme activity

ALT and AST enzymes were measured in blood of ICR mice, an animal model, in order to examine the efficacy of the hepatic hydrothermal extract and ethanol extract of the present invention.

Blood was collected using a heparin-treated capillary from the tail vein of the mouse, and the collected blood was left at room temperature for 1 hour, followed by centrifugation at 3000 rpm for 15 minutes to obtain serum. Store at 70 ° C. ALT was measured by the method of Reitman-Frankel using the Youngdong Pharmaceutical kit.

As a result, it was confirmed that the residual hot water extract and the ethanol extract decreased ALT and AST levels in a concentration-dependent manner (see FIG. 13).

< Experimental Example  12> From the Mouse Liver Tissue of Straw Extract TGF - β1  Activation inhibitory effect

To investigate the effect of inhibiting TGF-β1 expression, which is known to play a key role in the fibrosis of cells in liver tissues of ICR mice, which is an animal model, in order to investigate the efficacy of the hepatic hydrothermal extract and ethanol extract of the present invention RT-PCR was used for the measurement.

Liver tissue was collected and stored in a liquid nitrogen until it was frozen at -80 ° C. CDNA was synthesized using RNA-Bee (TEL-TEST, USA) using RNA extracted from image tissue using Maxime RT Premix (Oligo dT primer) kit (Intron Biotechnology, Korea). Quantitative polymerase chain reaction (qPCR) was measured using the Roter-Gene SYBR Green PCR reagent system (Qiagen, Germany). Each primer sequence information is as follows.

It was confirmed that the residual hot water extract and the ethanol extract inhibit the expression of transforming growth factor (TGF-β) gene in a concentration-dependent manner (see FIG. 14).

< Experimental Example  13> of twigs extract Non-alcoholic  Fatty liver metabolism regulation and mechanism study (In vivo)

In order to elucidate the mechanism of regulating fat synthase transcription in the hepatic efficacies of the remnant hydrothermal and ethanol extracts of the present invention, it is a transcription factor that regulates fat metabolism including fatty acid synthase (FAS) in liver tissue of animal model ICR mouse. Expression for sterol regulatory component binding protein-1 (SREBP-1) and AMPK that inhibits the expression of FAS and SREBP-1c was confirmed using Western blot.

Liver tissue was treated with IPH buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 100 μM PMSF), 1 μg / mL protease inhibitors (Luepetin, Aprotinin), 1 mM The protein was extracted by treatment with DTT. The extracted protein was transferred to a Hybond-P + polyvinylidene difluoride membrane (GE Healthcare, UK) after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Block the membrane with 5% fat-free milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 hours at room temperature, and the specific primary antibodies (SREBP-1c and phospho-AMPK) indicated. Incubated overnight at 4 ° C with TBS containing 1% BSA and 0.05% Tween-20. Membranes were again treated at room temperature for 2 hours with peroxidase-conjugated goat anti-mouse antibody or peroxidase-conjugated goat anti-rabbit antibody diluted 1: 3000, and 0.1% BSA and 0.1% Tween- After washing clean with 20 containing TBS, protein expression was confirmed using ECL plus (Amersham Biosciences). As a result, the residual hydrothermal and ethanol extracts inhibited the expression of sterol-response-component-binding protein 1c (SREBP-1c), fatty acid synthase (FAS), and AMP-activated protein kinase (AMPK) genes in a concentration-dependent manner. It was confirmed (see FIG. 15).

< Experimental Example  14> Suppression of Hedgehog signal that causes fibrosis of liver tissue (In vivo )

In order to confirm the effect of inhibiting liver fibrosis of the residual hot water extract and ethanol extract of the present invention, the expression of Gli family (Gli1 and Gli2), which are representative markers of Hedgehog signal in liver tissue of ICR mouse, which is an animal model, was examined. Confirmed using Western blot.

Liver tissue was treated with IPH buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 100 μM PMSF), 1 μg / mL protease inhibitors (Luepetin, Aprotinin), 1 mM The protein was extracted by treatment with DTT. The extracted protein was transferred to a Hybond-P + polyvinylidene difluoride membrane (GE Healthcare, UK) after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 5% fat-free milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 hours at room temperature to block membranes and 1% with the indicated primary antibodies (Gli1 and Gli2) Incubated overnight at 4 ° C. with BSA and TBS containing 0.05% Tween-20. The membranes were again treated with oxidase-conjugated goat anti-mouse antibody or peroxidase-conjugated goat anti-rabbit antibody diluted at 1: 3000 at room temperature for 2 hours, at 0.1% BSA and 0.1% Tween-20. After washing clean with containing TBS, protein expression was confirmed using ECL plus (Amersham Biosciences). As a result, it was confirmed that the residual hot water extract and the ethanol extract inhibit Gli1 and Gli2 expression in a concentration-dependent manner (see FIG. 16).

< Formulation example  1> Preparation of Pharmaceutical Formulations

<1-1> Preparation of Powder

2 g extract of Example 1

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

<1-2> Preparation of Tablet

100 mg of extract of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

100 mg of extract of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of Pills

1 g of extract of Example 1

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

<1-5> Preparation of Granules

150 mg of extract of Example 1

Soy extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

<1-6> Preparation of Injection

100 mg of extract of Example 1

Mannitol 180 mg

_{Na 2 HPO 4 · 12H 2 O} 26 ㎎

Distilled water 2974 mg

According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.

< Formulation example  2> Manufacture of food

<2-1> Preparation of Flour Food

0.5 to 5.0 parts by weight of the extract of Example 1 of the present invention was added to the flour and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> Preparation of Soups and Gravys

0.1 to 5.0 parts by weight of the extract of Example 1 of the present invention was added to soups and broth to prepare meat products for health promotion, soups of noodles and broths.

<2-3> Preparation of Ground Beef

10 parts by weight of the extract of Example 1 of the present invention was added to ground beef to prepare a ground beef for health promotion.

<2-4> Production of Dairy Products

5 to 10 parts by weight of the extract of Example 1 of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Preparation of Wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The extract of Example 1 of the present invention was concentrated under reduced pressure in a vacuum concentrator and dried by spraying and drying with a hot air dryer to grind the dried product to a particle size of 60 mesh using a grinder to obtain a dry powder.

The grains, seeds and the extract of Example 1 prepared above were combined and prepared in the following ratio.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Extract of Example 1 (3 parts by weight),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

< Formulation example  4> Manufacture of beverage

<4-1> Preparation of Health Beverage

Instantly mix the subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%) with 5 g of the extract of Example 1 of the present invention. After sterilization, it was prepared by packing it in a small packaging container such as a glass bottle or a plastic bottle.

<4-2> Preparation of Vegetable Juice

5 g of the extract of Example 1 of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<4-3> Preparation of Fruit Juice

1 g of the extract of Example 1 of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

Claims (10)

1. A bile acid modulator or FXR (panesoid X receptor) adjuvant comprising a remnant saponin made of any one of quilaic acid and gibsongenin represented by the following formula (1) or a remnant (Adenophora Radix) extract comprising the same as an active ingredient:

   [Formula 1]

   
2. The bile acid modulator or FXR anti-inflammatory agent according to claim 1, wherein the residue extract is a hot water extract or an organic solvent extract.
3. The method of claim 2, wherein the residual hydrothermal extract is

   Drying and grinding the residue in the shade to obtain residue powder;

   Adding 5-10 times water by volume to the obtained residue powder and extracting twice for 4 to 6 hours at a temperature of 90 to 100 ° C. to obtain a hydrothermal extract; And

   A bile acid regulator or FXR anti-tussive agent obtained by the method comprising the step of filtering or centrifuging the obtained hydrothermal extract to remove solids, concentrating and drying to obtain a residual hydrothermal extract powder.
4. The method of claim 2, wherein the residual organic solvent extract

   Drying and grinding the residue in the shade to obtain residue powder;

   To the obtained residue powder, an alcohol extract was obtained by adding 1 to 5 times C ₁ to C ₄ alcohol, or a mixture of C ₁ to C ₄ alcohol and water, and extracting twice at a temperature of 90 to 95 ° C. for 3 hours. Obtaining; And

   A bile acid regulator or FXR anti-tussive agent obtained by the method comprising the step of filtering or centrifuging the obtained alcohol extract to remove solids, concentrating and drying to obtain a residual alcohol extract powder.
5. The bile acid regulator or FXR anti-tussive agent according to claim 4, wherein the C ₁ to C ₄ alcohol is ethanol, the alcohol extract is an ethanol extract, and the residual alcohol extract powder is a residual ethanol extract powder.
6. The bile acid modulator or FXR anti-inflammatory agent according to claim 1, wherein the residue extract comprises 5 to 100% by weight.
7. A pharmaceutical composition for preventing and treating fatty liver disease, comprising a bile acid regulator or FXR anti-inflammatory agent according to any one of claims 1 to 6 as an active ingredient.
8. 8. The fatty liver according to claim 7, wherein the fatty liver disease is any one selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, obese fatty liver, and fatty liver disease group of diabetic fatty liver and primary biliary cirrhosis. Pharmaceutical compositions for preventing and treating diseases.
9. A functional food composition for preventing and improving fatty liver disease, comprising a bile acid regulator or FXR anti-inflammatory agent according to any one of claims 1 to 6 as an active ingredient.
10. The fatty liver according to claim 9, wherein the fatty liver disease is any one selected from the group consisting of alcoholic fatty liver, non-alcoholic fatty liver, obese fatty liver, and fatty liver disease group of diabetic fatty liver and primary biliary cirrhosis. Functional food composition for preventing and improving the disease.

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